Project Summary/Abstract Respiratory syncytial virus (RSV) causes significant lower respiratory tract illness (LRTI) in otherwise healthy children, and developing an effective prophylactic vaccine to prevent RSV disease in infants is a public and global health priority according to the World Health Organization. The specific goal of this research project is to generate, characterize, and prioritize, in the laboratory, a pipeline of novel live attenuated RSV vaccine candidates. We will also generate and assess live vaccine candidates against a related virus, human metapneumovirus (hMPV). Our long-term goal is to develop safe and efficacious RSV and hMPV vaccines for immunizing sero-negative infants, who are beyond the reach of protective maternal antibodies, against medically attended RSV and hMPV LRTI. Globally, there are 34 million new RSV infections annually with 3.4 million hospitalizations and 66,000 to 199,000 deaths. It was estimated that in the USA, 2 million children under the age of 5 required care for RSV infections annually and 78% are over the age of one year. hMPV is second to RSV as a major cause of infant LRTI. Vaccinating the birth cohort with a RSV vaccine that induces immunity similar to natural infection has been estimated to significantly reduce RSV hospitalizations and medical costs globally. A live attenuated vaccine generally mimics natural infection, and therein lays a challenge for RSV. On the one hand, epidemiology and natural history studies demonstrate that natural RSV infection results in protection of infants and children against severe RSV disease upon subsequent infection. On the other hand, natural RSV immunity is not considered robust or long lasting, so heavy attenuation of the natural virus led to poorly immunogenic live vaccines over the years. We recently published a RSV live vaccine candidate that was engineered to be highly attenuated in cotton rats yet retained immunogenicity similar to wild-type virus. Rather than attenuate virus replication per se, we codon-deoptimized or deleted non-essential RSV genes involved in virulence and immune modulation. This results in in vivo attenuation and enhanced immunogenicity. The attenuation phenotype was genetically stable because it is conferred by hundreds of mutations we introduced into the RSV genome. The other distinguishing characteristic is a novel fusion (F) protein that is enriched for the more immunogenic pre-F conformation. This grant expands these initial published findings into a vaccine discovery program to hone in an optimal RSV vaccine candidate and apply the approach to hMPV. Aims 1 and 2 will develop RSV live attenuated vaccine candidates based on optimizing virulence gene modifications and optimizing the F antigen. In Aim 3, we will utilize our RSV construct platform to generate chimeric RSV-hMPV strains as vaccine candidates against hMPV. The outcome of these studies will establish a new framework of balancing attenuation and potency and prioritize lead candidates for clinical development.